Title: Targeting of CDK6 in Myelofibrosis Project Summary/Abstract Myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are a group of hematologic malignancies characterized by excessive production of myeloid lineage cells. A somatic JAK2V617F mutation has been found in ~95% cases of PV and ~50-60% cases of ET and MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) also have been found in ET and MF. MF is the deadliest among MPNs. The median survival of patients with MF is ~5 years. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. Although Ruxolitinib treatment can reduce the splenomegaly and constitutional symptoms, it does not prevent fibrosis or produce disease remission in patients with MPN/MF. So, there is a critical need to identify new therapeutic target(s) and develop novel targeted therapies for MPN/MF. We have found that expression of CDK6 is significantly upregulated in mouse and human MPN hematopoietic cells/progenitors. CDK6 is a cyclin- dependent kinase which contributes to cell cycle progression and promotes cell growth. In preliminary studies, we have observed that knockdown of CDK6 significantly inhibits proliferation of hematopoietic cells expressing JAK2V617F. Furthermore, treatment with CDK6 inhibitor PD-0332991 (Palbociclib) markedly inhibits the growth of JAK2V617F-positive MPN cells. So, we hypothesize that CDK6 is an important target in MPN, and inhibition of CDK6 alone or in combination with JAK2 inhibition may be useful in treating MPN/MF. To test our hypothesis, we have proposed two specific aims. In Aim 1, we will determine the efficacy of the CDK6 inhibitor PD-0332991 alone or in combination with JAK2 inhibitor Ruxolitinib against cultured and primary MPN cells and animal models of MPN/MF. In Aim 2, we will determine the mechanism of inhibition of MPN by PD- 0332991 alone and/or PD-0332991/ Ruxolitinib drug combination. Our proposed studies will determine if targeting of CDK6 alone or in combination with JAK2 inhibition is effective against MPN/MF. Moreover, results from these pre-clinical studies will generate the supportive data for Phase I/II clinical trials involving PD- 0332991/ Ruxolitinib for treatment of MPN/MF.